Study of the Properties of Bearberry Leaf Extract As a Natural Antioxidant in Model Foods

antioxidants

Article Study of the Properties of Bearberry Leaf Extract as a Natural Antioxidant in Model Foods

Nurul Aini Mohd Azman 1,2, Maria Gabriela Gallego 1, Francisco Segovia 1, Sureena Abdullah 2, Shalyda Md Shaarani 2 and María Pilar Almajano Pablos 1,*

1 Chemical Engineering Department, Technical University of Catalonia, Avinguda Diagonal 647, Barcelona 08028, Spain; [email protected] (N.A.M.A.); [email protected] (M.G.G.); [email protected] (F.S.) 2 Chemical and Natural Resources Engineering Faculty, University Malaysia Pahang, Lebuhraya Tun Razak, Pahang 26300, Malaysia; [email protected] (S.A.); [email protected] (S.M.S.) * Correspondence: [email protected]; Tel.: +34-934-016-686; Fax: +34-934-017-150

Academic Editor: Dong Uk Ahn Received: 2 February 2016; Accepted: 24 March 2016; Published: 1 April 2016

Abstract: The common bearberry (Arctostaphylos uva-ursi L. Sprengel) is a ubiquitous procumbent evergreen shrub located throughout North America, Asia, and Europe. The fruits are almost tasteless but the plant contains a high concentration of active ingredients. The antioxidant activity of bearberry leaf extract in the 2,21-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation assay was 90.42 mmol Trolox equivalents/g dry weight (DW). The scavenging ability of the methanol extract of bearberry leaves against methoxy radicals generated in the Fenton reaction was measured via electron paramagnetic resonance. Lipid oxidation was retarded in an oil–water emulsion by adding 1 g/kg lyophilised bearberry leaf extract. Also, 1 g/kg of lyophilised bearberry leaf extract incorporated into a gelatin-based ﬁlm displayed high antioxidant activity to retard the degradation of lipids in muscle foods. The present results indicate the potential of bearberry leaf extract for use as a natural food antioxidant.

Keywords: bearberry leaves; scavenging activity; lipid oxidation; active packaging ﬁlm; antioxidant activity

1. Introduction Lipid oxidation in food causes serious problems that lead to short shelf lives and loss of nutritional quality [1]. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) have been used as antioxidants in many food products [2], but consumers have become concerned about possible toxicological effects and often prefer natural antioxidants for foods consumed as part of a healthy diet. Thus, many investigations have focused on identiﬁcation of novel antioxidants to be tested in model foods such as emulsions and incorporated into packaging ﬁlms. Natural antioxidants contain a high concentration of phenolic compounds and normally occur in fruits, vegetables, and herbs [3,4]. Bearberry (Arctostaphylos uva-ursi L. Sprengel) is a ubiquitous procumbent evergreen shrub located throughout North America, Asia, and Europe. The fruits are almost tasteless despite containing a high concentration of active ingredients in many commercial products [5]. The antioxidant potential of bearberry leaves (BL) has been studied by numerous chemical assays including reducing power assay, radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), liposome model, scavenging hydroxyl radicals (HO), and linoleic acid model system [6,7]. The main constituents of BL are the glycosides arbutin (5%–15%), methylarbutin (up to 4%), and small quantities of the free aglycones. Other constituents include ursolic acid, tannic acid, gallic acid,

Antioxidants 2016, 5, 11; doi:10.3390/antiox5020011 www.mdpi.com/journal/antioxidants Antioxidants 2016, 5, 11 2 of 11 p-coumaric acid, syringic acid, galloylarbutin, gallo-tannins, and flavonoids, notably glycosides of quercetin, kaempferol, and myricetin [8]. The traces of polyphenols in BL have made them promising candidates as potential protectors against lipid oxidation and biological ageing of tissues. Additionally, different phenolic compounds may act as antioxidants with varying efficiency in different food systems, which depend on their polarity and molecular characteristics. Studies on the efficacy of natural polyphenol derived from plants in food models are not new. Natural phenolics identified in many fruits, herbs, and vegetables used as antioxidants that retard the oxidative rancidity of foods in numerous food applications were summarized by Kiokias et al. [9]. They also critically discussed the recent new technology using multiple emulsions along with innovative applications of natural antioxidants in food [10]. Several studies of the antioxidant activity of BL at several concentrations have been conducted in a meat model [11,12] and these have successfully demonstrated the potential of bearberry to inhibit the degradation of lipids in pork. The potential of incorporating plants with phenolic compounds in emulsion was critically assessed by Kiokias et al. [13]. Among the tested flavonoid compounds, quercertin significantly reduced the oxidative deterioration of cottonseed oil-in-water emulsions. Quercetin is one of the phenolic compounds found in the BL. Hence, the potential effects of BL extract in oil-in-water emulsions are investigated in this study. Furthermore, there is also limited information on the utilization of the plant extracts incorporated into a film as active packaging. Therefore, this investigation aimed to: (a) investigate the potential antioxidant properties of BL extract using the Trolox Equivalent Antioxidant Capacity (TEAC) assay, and Electron Paramagnetic Resonance (EPR) scavenging activity; (b) demonstrate the ability of lyophilised BL to inhibit lipid deterioration directly in oil-water emulsions; and (c) study the effectiveness of gelatin-based film treated with lyophilised BL in retarding lipid oxidation in meat patties.

2. Experimental Section

2.1. Plant Material Commercial dried BL was kindly supplied by Pàmies Hortícoles (Balaguer, Spain), a registered herbal company. All reagents and solvents used were of analytical grade and obtained from Panreac (Barcelona, Spain) and Sigma Aldrich (Gillingham, England).

2.2. Extraction of BL Extract Dried BL was ﬁnely ground using a standard kitchen food processor. Ground BL was extracted with 50:50 (v/v) ethanol:water always in the ratio 1:20 (w/v). The extractions were performed in the dark at 4 ˘ 1 ˝C for 24 h, with constant stirring. The solutions of BL extract were recovered by ﬁltration using Whatman Filter paper, 0.45 µm. Part of the supernatant was taken for subsequent use to determine the antiradical capacity. The remaining supernatant was measured and the excess ethanol was removed under vacuum using a rotary evaporator (BUCHI RE111, Switzerland) and kept frozen at ´80 ˝C for 24 h. All extracts were dried in a freeze dryer (Unicryo MC2L ´60 ˝C, Munich, Germany) under vacuum at ´60 ˝C for 3 days to remove moisture. Finally, lyophilised BL were weighed to determine the soluble concentration (g/L) as described by Zhang et al. [14].

2.3. Determination of Total Phenolic Compound (TPC) The Folin–Ciocalteu method was used to determine the total phenolic content as described by Santas et al. [15].

2.4. Determination of Antioxidant Activity Using TEAC Assay The antioxidant capacity of BL was measured by a modiﬁed TEAC assay as described by Skowyra et al. [16], which was based on the method of Miller et al. [17]. Antioxidants 2016, 5, 11 3 of 11

2.5. Determination of Radical Scavenging Activity Assay Using Electron Paramagnetic Resonance (EPR) EPR radical scavenging activity was measured following the method of Azman et al. [18]. BL were extracted with MeOH in a ratio of 1:10 (w/v) and the soluble concentration of BL was determined as described in the procedure above. A spin-trapping reaction mixture consisted of 100 µL of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (35 mM), 50 µL of H2O2 (10 mM), and 50 µL BL extract solution at different concentrations. Ferulic acid was used as reference (0–20 g/L) and the control was pure MeOH. Finally, 50 µL of FeSO4 (2 mM) was added to the mixture. The ﬁnal solutions were transferred to a narrow quartz tube and introduced into the cavity of the EPR spectrometer. The spectrum was recorded for 10 min. X-band EPR spectra were recorded with a Bruker EMX-Plus 10/12 spectrometer (Bruker Española S.A., Madrid, Spain) under the following conditions: microwave frequency, 9.88 GHz; microwave power, 30.27 mW; centre ﬁeld, 3522.7 G; sweep width, 100 G; receiver gain, 5.02 ˆ 104; modulation frequency, 100 kHz; modulation amplitude, 1.86 G; time constant, 40.96 ms; conversion time, 203.0 ms.

2.6. Determination of Antioxidant Activity in Food Model

2.6.1. Removal of Tocopherols from Sunflower Oil Alumina was placed in an oven at 200 ˝C for 24 h, and then removed and allowed to cool in a desiccator until it reached room temperature. Sunflower oil was passed twice through the alumina in a column to remove the tocopherols as described by Yoshida et al. [19]. Finally, the filtered oil was stored at ´80 ˝C until use.

2.6.2. Preparation of Emulsion Oil-in-water emulsion was prepared using a method adapted from Azman et al. [20]. The ﬁnal samples were prepared using (1) control (no addition); (2) 0.2 g/kg BHA; or (3) 1 g/kg lyophilised BL. The emulsion for each sample was prepared in quadruplicate, obtaining a total of 12 samples and stored in the dark and allowed to oxidise at 37 ˝C. The pH of the samples was measured four times for each sample (pH meter GLP21, Crison Instruments, Barcelona, Spain) as a parameter to investigate its correlation with peroxide value (PV).

2.6.3. Determination of Peroxide Value (PV) The primary oxidation products were measured using peroxide value (PV) according to the thiocyanate method of the Association of Official Analytical Chemists (AOAC) 8195 [21]. Ferrous chloride solution was prepared in hydrochloric acid (1 M) with the addition of iron(II) chloride (2 mM, final concentration). Ammonium thiocyanate solution was prepared in water (2 mM, final concentration). The assay was performed with a drop of emulsion between 0.007 and 0.01 g, diluted with ethanol. From this solution, the required amount of sample, varying according to the degree of oxidation, was transferred into a cuvette and ethanol was added into the sample. Ferrous chloride and ammonium thiocyanate solutions were added, each in a proportion of 1.875% (v/v), final concentration. The absorbance was measured spectrophotometrically at λ = 500 nm. The results were expressed as meq hydroperoxides/kg of emulsion.

2.6.4. Preparation of Gelatin-Based Film with Antioxidant Coating The fabrication of gelatin-based film with antioxidant coating was adapted and characterized using the method of Bodini et al. [22]. During the cooling of the filmogenic solution after the solubilization of sorbitol, 1 g/kg of BL extract/gelatin was added. Fat and joint tissues were trimmed off lean meat (2000 g) and the meat was minced through 8-mm industrial plates. Then, the meat was moulded to a thickness of 1.5 cm. For each slice, films (5 ˆ 5 cm2) were placed on both sides, with either control film (no addition of antioxidant) or BL film (1 g/kg lyophilised BL). Control samples Antioxidants 2016, 5, 11 4 of 11 were prepared in the same manner except that the slices were not covered with any film. Subsequently, the samples were packed in polypropylene trays prior to storage at 4 ˝C for 12 days.

2.6.5. Thiobarbituric Acid Reacting Substances (TBARS) TBARS measurement was used to measure the extent of lipid oxidation during the storage period as described by Grau et al. [23]. Sample (1 g) was weighed in a tube and mixed with 3 ml/L aqueous EDTA. Then, the sample was immediately mixed with 5 mL of thiobarbituric acid reagent using an Ultra-Turrax (IKA, Germany) at 32,000 rpm speed for 2 min. All procedures were carried out in the dark and all samples were kept in ice. The mixture was incubated at 97 ˘ 1 ˝C in hot water for 10 min and shaken for 1 min during the process to form a homogeneous mixture. The liquid sample was recovered by ﬁltration (Whatman Filter paper, 0.45 µm) after the sample was cooled for 10 min. The absorbance value for each sample was measured at 531 nm using a spectrophotometer. The TBARS value was calculated from a Malondialdehyde (MDA) standard curve prepared with 1,1,3,3-tetraethoxypropane and analyzed by linear regression. All results were reported as mg malonaldehyde/kg sample.

2.7. Statistical Analysis A one-way analysis of variance (ANOVA) was performed using Minitab 16 software program (Addlink Software Cientiﬁco, Barcelona, Spain) (α = 0.05). The results were presented as mean values (n ě 3).

3. Results and Discussion

3.1. Extraction Yield, Total Phenolic Content (TPC) and Antioxidant Activity The extraction yield, total polyphenols (TPC), and antioxidant activity in extracts of bearberry leaves obtained with 50:50 v/v ethanol:water are shown in Table1. On average, 1.6 ˘ 0.01 g of extract pulp was recovered from 5 g of bearberry extract after 3 days freeze drying (p > 0.05).

Table 1. Extraction yield, polyphenol content and antioxidant activity of bearberry leaf extracts.

Activity Bearberry Extract Extraction Solvent 50:50 (v/v) EtOH:H2O* Extraction yield (%) 32.1% ˘ 0.03% Total phenolic content (mg GAE/g DW) 102.11 ˘ 7.12 TEAC (mmol of TE/g DW) 90.42 ˘ 1.83 *: Results are expressed as mean ˘ standard deviation (n = 3). Gallic acid equivalent (GAE), Trolox equivalent antioxidant activity (TEAC), Trolox equivalent (TE), dry weight (DW).

Pegg et al. (2005) reported that the total phenolic content of BL extract was 312 mg/g DW for 95% (v/v) ethanol extraction [6]. It was much higher than what we reported for 50% (v/v) ethanol extract. However, the total phenolic content value obtained from the BL water infusion was very low with 160.78 ˘ 2.84 g/kg sample [24]. The mixtures of alcohol and water was more efficient in extracting phenolic compounds and gave a better yield than water because some phenolic constituents do not dissolve in water. Meanwhile, the antioxidant activity of BL assessed using the TEAC method was 90.42 mmol of TE/g DW. A recent study reported the antioxidant activity of BL in the TEAC assay was 3.19 ˘ 0.01 mol of TE/kg sample following extraction by an infusion method which is much lower than our value [24]. The antioxidant activity of bearberry leaf achieved in the TEAC assay indicates the potency of the extract to scavenge the radical cation ABTS‚+ (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) generated in the assay. The use of several methods allows a more general assessment of the antioxidant properties of the plant. The variations of data were influenced by the sample preparation, type of extraction (solvent, temperature, etc.), selection of end-points, and method of expression of the results. Several studies have determined the antioxidant activity of BL extract using in vitro analysis. A few studies reported bearberry extract Antioxidants 2016, 5, 11 5 of 10 Antioxidants 2016, 5, 11 5 of 11 its derivatives, epigallocatechin gallate, epigallocatechin, and epicatechin. These catechins have a strongscavenging antioxidant ability capacity using the mainly DPPH linked radical to andtheir the radical ability scavenging of the extract activity to reduce [18]. ferric(III) ions to ferrous(II) ions using the FRAP (fluorescence recovery after photobleaching) method [15–17]. The 3.2 Analysis of Free Radical Activity Assays polyphenol constituents in the extract contribute the most to the antioxidant activity. The infusion of BLIn showed the present an abundance study, methanolic of phenolic extracts acid from components BL were at examined trace concentrations by EPR spectroscopy including for catechin their capacityand its derivatives, to act as radical epigallocatechin scavenger towards gallate, epigallocatechin, the methoxy radical and epicatechin.(CH3O•) generated These catechins by the Fenton have a reaction.strong antioxidant This method capacity was mainlyused to linked evaluate to their the radicalscavenging scavenging ability activityof plant [18 extracts]. for the free methoxy radical (CH3O•). Figure 1 shows the decrease in the EPR signal with increasing concentration3.2. Analysis of of Free BL Radical extract. Activity The free Assays radical scavenging activity of the extracts against methoxy (CH3OIn•) the radical present was study, investigated methanolic by a extractscompetitive from method BL were in examinedthe presence by EPRof DMPO spectroscopy as spin trap for theirand 3 •‚ recordedcapacity toby act the as spectrum radical scavenger generated towards by EPR the spectroscopy. methoxy radical The (CHCH 3OO )radical generated generated by the by Fenton the Fentonreaction. procedure This method has wasa relatively used to evaluateshort half-life, the scavenging which means ability it of must plant be extracts identified for the by free EPR methoxy as the ‚ 3 N stableradical nitroxide (CH3O ).adduct Figure with1 shows DMPO, the decreaseDMPO–OCH in the EPR(hyperfine signal splitting with increasing constants, concentration a = 13.9 G of and BL H 3 ‚ aextract. = 8.3 G). The This free stable radical DMPO–OCH scavenging compound activity of can the be extracts quantified against by the methoxy double (CH integration3O ) radical value was of theinvestigated signal from by EPR. a competitive The extract method containing in the antioxidants presence of at DMPO different as spin concentrations trap and recorded may compete by the ‚ withspectrum the spin generated trap DMPO by EPR in spectroscopy. the scavenging The CHof methoxy3O radical radicals. generated Thus, by thethe Fentoneffect decreases procedure the has amounta relatively of radical short half-life,adducts whichand, accordingly, means it must decreases be identified the intensity by EPR of as the the EPR stable signal. nitroxide The best adduct fit withwith intensity DMPO, DMPO–OCHof EPR signal3 exhibited(hyperfine a linear splitting function constants, (Figure aN 1),= 13.9given G by and Equation aH = 8.3 (1): G). This stable DMPO–OCH3 compound can bey quantified = −0.0694x by + 59.849; the double R2 = integration0.9871 value of the signal fromEPR. (1) The extract containing antioxidants at different concentrations may compete with the spin trap DMPO wherein the the scavenging x values ofwere methoxy in mg/L. radicals. Thus, the effect decreases the amount of radical adducts and, accordingly,The graph decreases indicates the intensitythe exponential of the EPR value signal. of Thethe bestsignal fit withof the intensity spectrum of EPR decreased signal exhibited as the amounta linear of function bearberry (Figure extract1), givenincreased. by Equation Azman (1):et al. demonstrated the scavenging ability of catechins with methoxy radical using this assay [18]. These catechins were also found in bearberry extract by Valjkovic et al. and these compoundsy “ ´ contributed0.0694x ` 59.849; to the Rabil2 “ity0.9871 to scavenge methoxy radical in this (1) assay [24]. Furthermore, BL scavenging ability has been previously reported by Amarowicz et al. usingwhere hydroxyl the x values free wereradicals in mg/L. (HO•) measured by EPR [11].

60 y = −0.0694x + 59.849 50 R² = 0.9871 40

20 Arbitrary Unit Arbitrary 10

0 0 200 400 600 800 Concentration (mg/L)

Figure 1. Variation in the area of the electron paramagnetic resonance (EPR) spectra of the radical

Figureadduct 1. DMPO–OCH Variation in3 thegenerated area of fromthe electron a solution paramagnetic of H2O2 (2 mM)resonance and FeSO (EPR)4 (0.04 spectra mM) of withthe radical DMPO adduct(14 mM) DMPO–OCH as spin trap3 generated in MeOH from as solvent. a solution The ofEPR H2O signal2 (2 mM) was and decreased FeSO4 (0.04 with mM) the with increased DMPO of (14concentration mM) as spin of thetrap BL in methanol MeOH as extracts. solvent. The The EPR EPR signal signal decreased was decreased at higher with antioxidant the increased activity. of concentration of the BL methanol extracts. The EPR signal decreased at higher antioxidant activity. The graph indicates the exponential value of the signal of the spectrum decreased as the amount 3.3. Antioxidant Effects in Stored o/w Emulsion of bearberry extract increased. Azman et al. demonstrated the scavenging ability of catechins with methoxyMethods radical have using been thisdeveloped assay [to18 ].understand These catechins the effect were of natural also found antioxidants in bearberry in model extract foods by suchValjkovic as emulsionset al. and and these active compounds film packaging. contributed Adding to the natural ability antioxidants to scavenge to methoxy food not radical only delays in this the oxidation process but also enhances the nutritional quality of the food through direct ingestion. Antioxidants 2016, 5, 11 6 of 11 assay [24]. Furthermore, BL scavenging ability has been previously reported by Amarowicz et al. using hydroxyl free radicals (HO‚) measured by EPR [11].

3.3. Antioxidant Effects in Stored o/w Emulsion Methods have been developed to understand the effect of natural antioxidants in model foods Antioxidants 2016, 5, 11 6 of 10 such as emulsions and active ﬁlm packaging. Adding natural antioxidants to food not only delays the oxidation process but also enhances the nutritional quality of the food through direct ingestion. In previous work, the effect of bearberry leaf extract in oil-water emulsion has not been described. In previous work, the effect of bearberry leaf extract in oil-water emulsion has not been described. A model emulsion was used to assess the deterioration of lipids at two stages of oxidation, which A model emulsion was used to assess the deterioration of lipids at two stages of oxidation, which were the primary oxidation products (Peroxide Value) and the secondary oxidation products were the primary oxidation products (Peroxide Value) and the secondary oxidation products (TBARS). (TBARS). In addition, the change in pH was monitored, since pH tends to fall during oxidation. In addition, the change in pH was monitored, since pH tends to fall during oxidation. The development of primary oxidation products was monitored by the evaluation of The development of primary oxidation products was monitored by the evaluation of hydroperoxide formation (PV) during storage (Figure 2). Primary degradation of lipids measured by PV hydroperoxide formation (PV) during storage (Figure2). Primary degradation of lipids measured by occurs due to the reaction between oxygen and unsaturated fatty acids thatform hydroperoxides. The PV occurs due to the reaction between oxygen and unsaturated fatty acids thatform hydroperoxides. induction time is defined as the time for samples to reach 10 meq hydroperoxides/kg of emulsion. This The induction time is deﬁned as the time for samples to reach 10 meq hydroperoxides/kg of emulsion. value can be used as a measure of the stability of emulsions. The limits of oxidation products in fat This value can be used as a measure of the stability of emulsions. The limits of oxidation products products (animal, plant and anhydrous) including margarine and fat preparations were set at <10 meq in fat products (animal, plant and anhydrous) including margarine and fat preparations were set at hydroperoxides/kg as a guarantee of the product quality [20]. When the peroxide value of the sample <10 meq hydroperoxides/kg as a guarantee of the product quality [20]. When the peroxide value of the is greater than 10 meq hydroperoxide/kg, the sample is in a highly oxidised state and starts to become sample is greater than 10 meq hydroperoxide/kg, the sample is in a highly oxidised state and starts to rancid. The PV value of the control emulsion increased rapidly, reaching more than 10 meq become rancid. The PV value of the control emulsion increased rapidly, reaching more than 10 meq hydroperoxide/kg after only 6 days (p < 0.05). The sample containing BL 1 g/kg reached the end of the hydroperoxide/kg after only 6 days (p < 0.05). The sample containing BL 1 g/kg reached the end of the induction time after 20 days while the BHT samples reached this state after 36 days of storage. induction time after 20 days while the BHT samples reached this state after 36 days of storage. Several Several studies investigated the effects of adding natural antioxidants to delay the lipid deterioration studies investigated the effects of adding natural antioxidants to delay the lipid deterioration in food in food model emulsions. Skowyra et al. [16] found that an emulsion containing 48 µg/mL of Tara extract model emulsions. Skowyra et al. [16] found that an emulsion containing 48 µg/mL of Tara extract took took 13 days to reach more than 10 meq hydroperoxide/kg, and Roedig–Penman and Gordon [25] 13 days to reach more than 10 meq hydroperoxide/kg, and Roedig–Penman and Gordon [25] reported reported that an emulsion containing green tea extract required eight days to reach the end of the that an emulsion containing green tea extract required eight days to reach the end of the induction time. induction time. Emulsion containing 100 mg/L of rosemary and thyme extract displayed low PV Emulsion containing 100 mg/L of rosemary and thyme extract displayed low PV which remained which remained below 10 for 25 days of storage [25]. below 10 for 25 days of storage [25].

70 Control 60 BHA 50 Bearberry lyophilise 40

30 emulsión)

PV (meq hidroperoxide / kg PV (meq hidroperoxide 10

0 0 1020304050 Day

Figure 2. Change in peroxide value over time stored at 37 ˝C. Each value is expressed as mean (n = 3). Figure 2. Change in peroxide value over time stored at 37 °C. Each value is expressed as mean (n = 3).

PrimaryPrimary oxidationoxidation occursoccurs rapidlyrapidly inin thethe fatfat phasephase ofof thethe productproduct duedue toto thethe formationformation ofof highlyhighly unstableunstable hydroperoxideshydroperoxides thatthat breakbreak downdown easily.easily. ThisThis processprocess resultsresults inin thethe formationformation ofof ketones,ketones, epoxides,epoxides, oror organicorganic acidsacids whichwhich areare acidic, acidic, and and leads leads to to changes changes in in the the pH pH [ 16[16].]. FigureFigure3 3shows shows that that thethe pHpH valuevalue droppeddropped overover time,time, andand thisthis changechange waswas inverselyinversely proportionalproportional to the increase of PV. By comparison, emulsion containing BHA showed a significant pH difference from the pH values for BL and the control sample (p < 0.05). BL samples remained higher than pH 6 for 10 days of storage and the pH declined gradually until 40 days. A number of researchers suggested a positive effect of pH on oxidation rate which was influenced by natural antioxidants [16,17,26]. Pehlivan et al. [27] reported that edible vegetable oils contained heavy metals such as iron up to 0.2 mg/kg oil. The levels of some metal compounds, if high enough, will promote oxidation which affects the pH [28]. Furthermore, the redox state of metals and the activity, solubility, stability, and chelation capacity of antioxidants are among the parameters that affect the rate of change of pH in oil emulsions [29]. Antioxidants 2016, 5, 11 7 of 11

By comparison, emulsion containing BHA showed a signiﬁcant pH difference from the pH values for AntioxidantsBL and the 2016 control, 5, 11 sample (p < 0.05). BL samples remained higher than pH 6 for 10 days of storage7 of 10 and the pH declined gradually until 40 days.

12 Control

10 BHA

8 Bearberry Lyophilise

6 pH 4

0 0 1020304050 Day

Figure 3. Change of pH over time stored at 37 ˝C. Each value is expressed as mean (n = 3). Figure 3. Change of pH over time stored at 37 °C. Each value is expressed as mean (n = 3).

AnalysisA number of ofoxidation researchers in the suggested emulsions a positive was extended effect of by pH measuring on oxidation a secondary rate which oxidation was inﬂuenced product inby the natural emulsion antioxidants using the [ 16TBAR,17,26S ].method Pehlivan (Figureet al. 4).[27 Malondiald] reportedehyde that edible (MDA) vegetable compounds oils containedproduced areheavy responsible metals such for asthe iron alteration up to 0.2 of mg/kgflavor, oil.rancid The odor, levels and of somethe undesirable metal compounds, taste in iffoods high [30].The enough, acceptablewill promote limits oxidation of TBARS which value affects in fat theproducts pH [28 wa].s Furthermore,set at 1.0 mg malondialdehyde/kg the redox state of metals [31]. Figure and the 4 showsactivity, the solubility, secondary stability, oxidation andchelation products capacity monitore ofd antioxidants by TBARS areassay. among Control the parameterssample experienced that affect TBARSthe rate value of change of greater of pH than in oil 4.0 emulsions mg malondialdehyde/kg [29]. (p < 0.05) on the 3 weeks storages compare to samplesAnalysis treated of oxidation with antioxidant in the emulsions with value was extended of TBARS by that measuring less than a secondary1 mg malondialdehyde/kg. oxidation product Overin the the emulsion 25 days using of thestorage, TBARS samples method with (Figure antioxidants4). Malondialdehyde had a TBARS (MDA) value compounds of approximately produced 1are mg responsible malondialdehyde/kg for the alteration sample, ofwhich ﬂavor, may rancid not change odor, andthe physical the undesirable and visual taste properties in foods of [the30]. product.The acceptable Emulsion limits treated of TBARS with BHA value exhibited in fat productsthe lowest was TBARS set at value 1.0 mg throughout malondialdehyde/kg the storage period [31]. andFigure samples4 shows with the bearberry secondary had TBARS oxidation values products of lower monitored than 2 mg bymalondialdehyde/kg TBARS assay. Control (p < 0.05). sample experienced TBARS14 value of greater than 4.0 mg malondialdehyde/kg (p < 0.05) on the 3 weeks storages compare to samplesControl treated with antioxidant with value of TBARS that less than 1 mg malondialdehyde/kg.12 Over the 25 days of storage, samples with antioxidants had a TBARS value BHA of approximately 1 mg malondialdehyde/kg sample, which may not change the physical and visual properties10 of the product.Bearberry Emulsion Lyophilisate treated with BHA exhibited the lowest TBARS value throughout the storage8 period and samples with bearberry had TBARS values of lower than 2 mg malondialdehyde/kg (p < 0.05). TBARS emusion 6 4

(mg malondialdehyde / (mg malondialdehyde kg 2 0 21 25 28 32 35 38 Day

Figure 4. Change of TBARS over time during storage at 37 °C. Each value is expressed as mean (n = 3).

Many findings demonstrated that the efficiency of natural plant extracts as antioxidants in various concentrations applied possibly depends on the variation in the system tested and initial plant extraction. The active properties of BL was reported several times by Amarowicz et al. [11,32]. Quercetin, one of the main compounds found in the BL extract, showed positive effects against lipid Antioxidants 2016, 5, 11 7 of 10

12 Control

10 BHA

8 Bearberry Lyophilise

6 pH 4

0 0 1020304050 Day

Figure 3. Change of pH over time stored at 37 °C. Each value is expressed as mean (n = 3).

Analysis of oxidation in the emulsions was extended by measuring a secondary oxidation product in the emulsion using the TBARS method (Figure 4). Malondialdehyde (MDA) compounds produced are responsible for the alteration of flavor, rancid odor, and the undesirable taste in foods [30].The acceptable limits of TBARS value in fat products was set at 1.0 mg malondialdehyde/kg [31]. Figure 4 shows the secondary oxidation products monitored by TBARS assay. Control sample experienced TBARS value of greater than 4.0 mg malondialdehyde/kg (p < 0.05) on the 3 weeks storages compare to samples treated with antioxidant with value of TBARS that less than 1 mg malondialdehyde/kg. Over the 25 days of storage, samples with antioxidants had a TBARS value of approximately 1 mg malondialdehyde/kg sample, which may not change the physical and visual properties of the Antioxidants 2016, 5, 11 8 of 11 product. Emulsion treated with BHA exhibited the lowest TBARS value throughout the storage period and samples with bearberry had TBARS values of lower than 2 mg malondialdehyde/kg (p < 0.05). 14 Control 12 BHA

10 Bearberry Lyophilisate 8 TBARS emusion 6 4

(mg malondialdehyde / (mg malondialdehyde kg 2 0 21 25 28 32 35 38 Day

FigureFigure 4. 4. ChangeChange of of TBARS TBARS over time duringduring storagestorage atat 3737˝ °C.C. EachEach valuevalue isis expressedexpressed as as mean mean ( n(n= = 3). 3).

Many findings demonstrated that the efficiency of natural plant extracts as antioxidants in Many findings demonstrated that the efficiency of natural plant extracts as antioxidants in various concentrations applied possibly depends on the variation in the system tested and initial plant various concentrations applied possibly depends on the variation in the system tested and initial extraction. The active properties of BL was reported several times by Amarowicz et al. [11,32]. plant extraction. The active properties of BL was reported several times by Amarowicz et al. [11,32]. Quercetin, one of the main compounds found in the BL extract, showed positive effects against lipid Quercetin, one of the main compounds found in the BL extract, showed positive effects against lipid deterioration in oil-in-water emulsion [9]. The leaves consist of an abundance of phenolic compounds, which may contributed to the antioxidant properties, reducing power, and antiradical properties as described in much of the literature [7,11,32]. The antioxidant activity of phenolic linked with phenolic compounds is related to the hydroxyl group linked to the aromatic ring, which is capable of donating hydrogen atoms and neutralizing free radicals. This mechanism prevents further propagation of lipid oxidation, which can be measured by the TBARS method [33]. The behavior of BL as a natural antioxidant in a food model was reported in the literature [12,34]. Pegg et al. (2005) [9] reported 200 ppm of bearberry extract inhibited TBARS formation in cooked pork patties after seven days of storage. Meanwhile, Carpenter et al. [12] demonstrated that the addition of BL impeded lipid oxidation in raw pork patties for 9–12 days of refrigerated storage relative to control. To the best of our knowledge, this is the first report of the inhibitory effect of BL using oil-in-water emulsion.

3.4. Antioxidant Effects in Active Film Packaging with Bearberry Coating The TBARS index (Figure5) revealed that secondary oxidation of the control beef patties increased progressively as storage advanced. Meat patties without any film on the patties (control sample) experienced the highest TBARS values and the values were significantly different than those of all other samples. Coating with gelatin-based film enriched with BL extract lowered the oxidation rate significantly throughout the storage period (p < 0.05). At the end of the storage time, patties stored under the gelatin film with BL contained only 0.17 mg malondialdehyde/kg sample compared to the control TBARS value of 0.86 mg malondialdehyde/kg sample. There are very few reports dealing with the effects of edible gelatin-based films containing natural plant extracts. Published studies of gelatin-based films focused on the physical, chemical, and mechanical properties of the film [20–27]. However, there are only a few studies reported the antioxidant effects of gelatin film treated with various natural antioxidants. Recently, several reports proposed that the combination of natural plant with antioxidant with gelatin film not only to improve its physical properties also delays the oxidation process within the food packaging [34,35]. This study reported the preliminary results of the effects of bearberry extract incorporated into gelatin film which successfully delayed oxidation in a muscle food. Antioxidants 2016, 5, 11 8 of 10 deterioration in oil-in-water emulsion [9]. The leaves consist of an abundance of phenolic compounds, which may contributed to the antioxidant properties, reducing power, and antiradical properties as described in much of the literature [7,11,32]. The antioxidant activity of phenolic linked with phenolic compounds is related to the hydroxyl group linked to the aromatic ring, which is capable of donating hydrogen atoms and neutralizing free radicals. This mechanism prevents further propagation of lipid oxidation, which can be measured by the TBARS method [33]. The behavior of BL as a natural antioxidant in a food model was reported in the literature [12,34]. Pegg et al. (2005) [9] reported 200 ppm of bearberry extract inhibited TBARS formation in cooked pork patties after seven days of storage. Meanwhile, Carpenter et al. [12] demonstrated that the addition of BL impeded lipid oxidation in raw pork patties for 9–12 days of refrigerated storage relative to control. To the best of our knowledge, this is the first report of the inhibitory effect of BL using oil-in-water emulsion.

1.0

0.8 Control sample 0.6 Control film

TBARS 0.4 Bearberry lyophilisate

0.2 (mg malondialdehyde/kg sample

0.0 02468 Day

Figure 5. Changes in TBARS values (mg malondialdehyde/kg sample) of control and sample Figurecontaining 5. Changes BL extract in TBARS during values seven days(mg ofmalondialdehyde/kg storage at 4 ˘ 1 ˝C sample) without of light. control Each and sample sample was containingmeasured BL in triplicateextract during and the se averageven days standard of storage deviation at 4 ± for 1 each°C without sample waslight. less Each than sample 5%. was measured in triplicate and the average standard deviation for each sample was less than 5%. 4. Conclusions In conclusion, BL extract showed a positive effect due to its antioxidant activity and scavenging ability, which delayed lipid oxidation in an emulsion and when used as an active component in gelatin film packaging. BL extract contained a high concentration of phenolic compounds and good antioxidant activity when assessed by total phenolic content and TEAC assay, respectively. The methanol extract showed scavenging ability against methoxy radicals generated by the Fenton reaction when assessed by EPR. Lyophilised BL (0.1% w/w) was applied as an antioxidant in an emulsion food model and significantly inhibited lipid oxidation during 20 days of storage. A preliminary study of the effect of gelatin-based film coated with BL extract showed that it significantly delayed degradation of lipids in meat patties (p < 0.05). Therefore, this study confirmed that bearberry leaves can be used as a source of antioxidants with potential use for the food industry.

Acknowledgments: The authors acknowledge the special fund from The Technical University of Catalonia, University Malaysia Pahang and Malaysia Government for the financial support of this research. Author Contributions: Nurul Aini Mohd Azman wrote the first draft; Maria Gabriela Gallego, Francisco Segovia, Sureena Abdullah, Shalyda Md Shaarani and Nurul Aini Mohd Azman did the experimental section; and María Pilar Almajano Pablos has been the supervisor and manager of the whole process. Conflicts of Interest: The authors declare no conflict of interest.

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